Rapid sex chromosomal chimerism analysis in heterosexual twin female calves by Loop-mediated Isothermal Amplification

We attempted to apply an embryo sexing kit with Loop-mediated Isothermal Amplification (LAMP) to sex chromosomal chimerism analysis in heterosexual twin female calves. Peripheral blood was used for the amplification of male-specific DNA, derived from XY leukocytes. When blood samples were diluted 1:1000 in LAMP reaction mixture, hemoglobin or blood coagulation did not influence the turbidity measurement of the reaction mixture for detection of amplified DNA. This procedure detected the existence of XY leukocytes of 0.01% in female blood. Furthermore, all heterosexual twin female calves, bearing sex chromosomal chimerism based on karyotyping and PCR, showed male-specific DNA from peripheral blood by LAMP. These results indicated that the embryo sexing kit with LAMP was available for sensitive detection of sex chromosomal chimerism. This procedure made it possible to detect easily Y-chromosome specific DNA in a short interval compared with PCR, and was convenient for field application of freemartin diagnosis.     

Birth of a Holstein freemartin calf co-twinned to a schistosomus reflexus fetus

An unusual case of a live Holstein freemartin calf co-twinned with schistosomus reflexus fetus is presented here. Delivery of the schistosomus reflexus was achieved by fetotomy 24 h after manual delivery of a live heifer calf. The dam subsequently experienced concurrent metritis and left displacement of the abomasum; however, she conceived following insemination 85 d post partum. Cytogenetic examination of the live heifer using lymphocyte culture and cutaneous fibroblast cell culture failed to demonstrate chromosomal chimerism, whereas polymerase chain reaction (PCR) detected the presence of the bovine Y-chromosome marker BRY-1. Low concentrations of testosterone, estradiol and progesterone at 3, 6, 24 and 48 h after administration of hCG were detected in the serum of the freemartin heifer. Genetic, hormonal, histological and clinical findings established the live female co-twin calf was a freemartin, an abnormality of phenotypic sex. Failure to detect any significant peripheral concentrations of androgen supports the hypothesis that masculinization of the freemartin reproductive tract arises from diffusion of androgen and possibly other substances from the male co-twin rather than from endogenous production of androgen within the freemartin. This report documents that the freemartin condition can be induced by a male fetus with severe developmental abnormalities.     

Cytogenetic observations on XX/XY chimaeras and a reassessement of the evidence for germ cell chimaerism in heterosexual twin cattle and marmosets.

Testicular preparations were obtained from 7 bulls, twins of freemartins, and 1 male marmoset, all proved XX/XY chimaeras. X and Y sex chromosomes were confidently identified in nearly all the 87 spermatogonia at mitotic metaphase and 1052 primary spermatocytes at diakinesis-metaphase examined: no cell was identified as containing two X chromosomes. The germ cell chimaerism previously reported in these species is therefore not confirmed. Cultures grown from presumptive somatic these species is therefore not confirmed. Cultures grown from presumptive somatic cells in the testes of two of the bulls yielded 248 identifiable mitotic spreads, all XY-type; cultures from the gonads of their freemartin twins yielded 442 mitotic spreads, all XX-type. Direct preparations from one freemartin gonad, however, yielded 3 XY mitotic spreads out of 18 examined. The conflicting evidence concerning germ cell chimaerism in cattle and marmosets is discussed, particularly in relation to reports of XX/XY bulls that have sired a great excess of daughters. The possibility that XX germ cells contributed to the functional spermatozoa of these bulls is not favoured by present information, but is not excluded.     

60,XY/60,XX chimerism in the germ cell line of mature bulls born in heterosexual twinning

According to present knowledge there is a germ cell chimerism (XY/XX) in young bulls born in heterosexual twinning due to exchange of primordial germ cells in embryonic life. These germ cells were believed to have been eliminated in the young bull. Two-color fluorescence in situ hybridization (FISH) identification of the sex chromosomes by biotinylated and digoxygenin labeled probes have been used. The material consisted of three bulls born in heterosexual twinning. The results obtained indicated that even mature bulls (more than two years old) demonstrate spermatogonial chimerism. Several authors state that the bulls with blood cell chimerism, originating from dizygous twinning, are characterized by decreased fertility. Changes of the sex ratio of offspring due to proliferation of the female cells have also been proposed. The present observations should give a renewed interest in checking the possibility of survival and differentiation of germ cells from the female partner in the germ cell lines.     

Birds bias offspring sex ratio in response to livestock grazing

Livestock grazing, which has a large influence on habitat structure, is associated with the widespread decline of various bird species across the world, yet there are few experimental studies that investigate how grazing pressure influences avian reproduction. We manipulated grazing pressure using a replicated field experiment, and found that the offspring sex ratio of a common upland passerine, the meadow pipit Anthus pratensis, varied significantly between grazing treatments. The proportion of sons was lowest in the ungrazed and intensively grazed treatments and highest in treatments grazed at low intensity (by sheep, or a mixture of sheep and cattle). This response was not related to maternal body condition. These results demonstrate the sensitivity of avian reproductive biology to variation in local conditions, and support growing evidence that too much grazing, or the complete removal of livestock from upland areas, is detrimental for common breeding birds.     

Freemartinism and FecX allele determination in replacement ewes of the Rasa Aragonesa sheep breed by duplex PCR

A new naturally occurring mutation in the fecundity gene BMP15 in the Rasa Aragonesa sheep breed (Ovis aries) has been found to affect prolificacy. This mutation (FecX^R allele) is a deletion of 17 base pairs that leads to an altered amino acid sequence, and this alteration increases prolificacy in heterozygous ewes but causes sterility in homozygous ewes. Selection of repository lambs with the FecX^R allele increases rates of twins and multiple lambing and thereby also increases the probability of lambing freemartins that will become sterile. In this sense, an accurate, reliable, and quick method was developed by duplex polymerase chain reaction (PCR) for sex, amplifying an ovine-specific Y chromosome repetitive fragment, and BMP15 genotype determination in replacement ewe lambs. The BMP15 fragment served as an internal control of the amplification and detected the FecX^R allele, avoiding a false negative and then a mistake in freemartin detection. This assay uncovered 6 freemartin females among 195 replacement ewes from 7 different commercial flocks and 1 experimental flock. Furthermore, 1554 rams from 64 commercial flocks were also analyzed to identify FecX^R rams. This analysis identified 103 rams hemizygous for the FecX^R allele and 1 heterozygous ram. Because this gene is located on the X chromosome, this heterozygous animal is a freemartin ram that is co-amplifying the DNA from XX and XY lymphocytes. These results confirm the usefulness of this multiplex PCR assay for detecting phenotypically sexed females, freemartins, and the BMP15 genotype to detect highly prolific ewes in commercial flocks and to assist breeders in selection of repository lambs.     

Direct observation of hematopoietic progenitor chimerism in fetal freemartin cattle

Background

Cattle twins are well known as blood chimeras. However, chimerism in the actual hematopoietic progenitor compartment has not been directly investigated. Here, we analyzed fetal liver of chimeric freemartin cattle by combining a new anti-bovine CD34 antibody and Y-chromosome specific in situ hybridization.

Results

Bull-derived CD34⁺ cells were detected in the liver of the female sibling (freemartin) at 60 days gestation. The level of bull-derived CD34⁺ cells was lower in the freemartin than in its male siblings. Bull (Y⁺) and cow hematopoietic cells often occurred in separate clusters. Around clusters of Y⁺CD34⁺ cells, Y⁺CD34^- cells were typically observed. The thymi were also strongly chimeric at 60 days of gestation.

Conclusion

The fetal freemartin liver contains clusters of bull-derived hematopoietic progenitors, suggesting clonal expansion and differentiation. Even the roots of the hematopoietic system in cattle twins are thus strongly chimeric from the early stages of fetal development. However, the hematopoietic seeding of fetal liver apparently started already before the onset of functional vascular anastomosis.     

A fast, convenient diagnosis of the bovine freemartin syndrome using polymerase chain reaction

To establish the polymerase chain reaction (PCR) method for detecting the XY cells in cases suspected to have the bovine freemartin syndrome, a PCR reaction test was conducted on blood from a normal bull diluted in blood from a normal cow. From the results obtained, it was shown that the Y-specific sequence was detectable down to a concentration of 0.1%. Various types of the bovine freemartin syndrome, which occurs in heterosexual twins, single-born sterile heifers, and heifers born with Acardius amorphus, were examined by the chromosome analysis and the PCR method. The Y-specific sequence was detected in all 26 cases that showed chromosome chimerism but which was absent in the 5 cases without a chimerism. The PCR method was found to be effective and convenient for quickly diagnosing the various types of bovine freemartin syndrome.     

Maternal eNOS deficiency determines a fatty liver phenotype of the offspring in a sex dependent manner

Maternal environmental factors can impact on the phenotype of the offspring via the induction of epigenetic adaptive mechanisms. The advanced fetal programming hypothesis proposes that maternal genetic variants may influence the offspring's phenotype indirectly via epigenetic modification, despite the absence of a primary genetic defect. To test this hypothesis, heterozygous female eNOS knockout mice and wild type mice were bred with male wild type mice. We then assessed the impact of maternal eNOS deficiency on the liver phenotype of wild type offspring. Birth weight of male wild type offspring born to female heterozygous eNOS knockout mice was reduced compared to offspring of wild type mice. Moreover, the offspring displayed a sex specific liver phenotype, with an increased liver weight, due to steatosis. This was accompanied by sex specific differences in expression and DNA methylation of distinct genes. Liver global DNA methylation was significantly enhanced in both male and female offspring. Also, hepatic parameters of carbohydrate metabolism were reduced in male and female offspring. In addition, male mice displayed reductions in various amino acids in the liver. Maternal genetic alterations, such as partial deletion of the eNOS gene, can affect liver metabolism of wild type offspring without transmission of the intrinsic defect. This occurs in a sex specific way, with more detrimental effects in females. This finding demonstrates that a maternal genetic defect can epigenetically alter the phenotype of the offspring, without inheritance of the defect itself. Importantly, these acquired epigenetic phenotypic changes can persist into adulthood.     

No evidence for sex biases in milk macronutrients,energy, or breastfeeding frequency in a sample of filipino mothers

Maternal reproductive investment includes both the energetic costs of gestation and lactation. For most humans, the metabolic costs of lactation will exceed those of gestation. Mothers must balance reproductive investment in any single offspring against future reproductive potential. Among mammals broadly, mothers may differentially invest in offspring based on sex and maternal condition provided such differences investment influence future offspring reproductive success. For humans, there has been considerable debate if there are physiological differences in maternal investment by offspring sex. Two recent studies have suggested that milk composition differs by infant sex, with male infants receiving milk containing higher fat and energy; prior human studies have not reported sex‐based differences in milk composition. This study investigates offspring sex‐based differences in milk macronutrients, milk energy, and nursing frequency (per 24 h) in a sample of 103 Filipino mothers nursing infants less than 18 months of age. We found no differences in milk composition by infant sex. There were no significant differences in milk composition of mothers nursing first‐born versus later‐born sons or daughters or between high‐ and low‐income mothers nursing daughters or sons. Nursing frequency also showed no significant differences by offspring sex, sex by birth order, or sex by maternal economic status. In the Cebu sample, there is no support for sex‐based differences in reproductive investment during lactation as indexed by milk composition or nursing frequency. Further investigation in other populations is necessary to evaluate the potential for sex‐based differences in milk composition among humans. Am J Phys Anthropol 152:209–216, 2013. © 2013 Wiley Periodicals, Inc.     

Behavioral characteristics of freemartins administered estradiol, estrone, testosterone, and dihydrotestosterone

Eighteen genetic females born co-twin with males and diagnosed as being sterile intersexes (freemartins) were studied from birth to 79 weeks of age. Testosterone (T) and estrone (EI) were administered in Silastic capsules of two groups from birth to 50 weeks of age and other animals were left untreated. At 50 weeks the two treated groups had larger implants installed and the untreated animals were assigned to a new estrone (EII) and estradiol (E2) treatment. Later a dihydrotestosterone (DHT) group was formed in comparison with new E2 and testosterone propionate-enanthate (TP-TE) groups, plus untreated controls. Vulvar interest, Flehmen lip curl, mounting, and agonistic behavior were recorded daily for 30 min while animals were allowed social interaction. Agonistic behavior, interest in the genital area, and mounting were induced or stimulated by T, TP-TE, and E2, but not by DHT or estrone (EI or EII). Also, only animals in the T, TP-TE, and E2 groups induced to mount displayed the standing type of behavioral estrus. Flehmen lip curl was stimulated only by T or TP-TE. The evidence is interpreted to indicate that T, per se, evokes the lip curl, but it probably stimulates other responses at the neural level by conversion to E2. Also, the freemartin response, the response of castrates to steroid hormones, and current knowledge of circulating steroid hormones in male and female cattle could be interpreted to indicate that the neural tissue responsible for sexual behavior in both sexes of this species may respond similarly in several respects.     

Effect of reproductive compensation and the desire to have male offspring on the incidence of a sex-linked lethal disease.

The effects of reproductive compensation on an X-linked recessive lethal are examined. Complete compensation without regard to the sex of the offspring increases the incidence of female carriers by a factor of 1.5, and of affected males by 1.33. However, if families reproduce until they have a healthy male offspring, the incidence of the X-linked lethal can be increased two or three orders of magnitude. Even only 1% of the population reproducing until a male is born can inflate the incidence of the disease by an order of magnitude, provided this pattern of family planning is culturally inherited. Similarly, reproducing until there is at least one child of each sex increases the incidence of an X-linked lethal. The impact of these types of sex-biased family planning on the fraction of new mutants among affect males is discussed.     

Freemartinism among singleton bovine females born from multiple embryo transfer

Heterosexual chimerism among singleton females produced by multiple nonsexed embryo transfer (MNET singleton females) was investigated using chromosome typing and PCR (polymerase chain reaction)-amplification of male-specific DNA (msDNA). Of the 22 animals tested, 21 were classified as normal by both methods (i.e., showing no male cells among 100 metaphase spreads in chromosome typing and being msDNA negative in PCR). No morphological abnormalities of the genital organs were observed among 19 MNET single females. One MNET singleton female was, however, classified as a freemartin by PCR (male-specific DNA positive), but it was classified as normal cytogenetically. This individual probably had a low degree of heterosexual chimerism, and it seems that the chimerism derived from MNET was difficult to diagnose by chromosome typing, although it was detectable by PCR. The genital organs of this individual (15-mo-old Aberdeen Angus) were normal in form (both external and internal) and size. However, a very small structure, resembling seminiferous tubule, was found in the left ovary. It may be concluded that most MNET singleton females are expected to have normal reproductive function.     

True hermaphroditism in a wild sheep: A clinical report

Intersexuality in sheep is rare, with the freemartin anomaly being the most common. We describe here a true hermaphrodite in a wild sheep. An F(1) wild sheep ewe of Argali-mouflon X Mexican desert bighorn breeding was bred to an F(1) ram of the same breeding. A single lamb was born with the external appearance of a normal female. The lamb grew faster than its female cohorts, and by 6 months of age exhibited the aggressive behavior, size, coloration and horn development associated with males. Phenotypically, the intersex had female external genitalia with an enlarged clitoris. A human chorionic gonadotrophin (hCG) response test was performed when the intersex was 1-year-old and serum testosterone, progesterone and estradiol levels were compared to the response of a normal female and male of similar age and breeding. An exploratory celiotomy revealed two gonadal-like structures associated with a female reproductive tract. Histopathology of the structures revealed spermatogenically inactive testicular vessels and ovarian tissue with primary follicles. The reproductive tract was complete with two uterine horns and a cervix. The intersexuality is attributed to an XX/XXY mosaic.     

No seasonal sex-ratio shift despite sex-specific fitness returns of hatching date in a lizard with genotypic sex determination

Sex allocation theory predicts that mothers should adjust their sex-specific reproductive investment in relation to the predicted fitness returns from sons versus daughters. Sex allocation theory has proved to be successful in some invertebrate taxa but data on vertebrates often fail to show the predicted shift in sex ratio or sex-specific resource investment. This is likely to be partly explained by simplistic assumptions of vertebrate life-history and mechanistic constraints, but also because the fundamental assumption of sex-specific fitness return on investment is rarely supported by empirical data. In short-lived species, the time of hatching or parturition can have a strong impact on the age and size at maturity. Thus, if selection favors adult sexual-size dimorphism, females can maximize their fitness by adjusting offspring sex over the reproductive season. We show that in mallee dragons, Ctenophorus fordi, date of hatching is positively related to female reproductive output but has little, if any, effect on male reproductive success, suggesting selection for a seasonal shift in offspring sex ratio. We used a combination of field and laboratory data collected over two years to test if female dragons adjust their sex allocation over the season to ensure an adaptive match between time of hatching and offspring sex. Contrary to our predictions, we found no effect of laying date on sex ratio, nor did we find any evidence for within-female between-clutch sex-ratio adjustment. Furthermore, there was no differential resource investment into male and female offspring within or between clutches and sex ratios did not correlate with female condition or any partner traits. Consequently, despite evidence for selection for a seasonal sex-ratio shift, female mallee dragons do not seem to exercise any control over sex determination. The results are discussed in relation to potential constraints on sex-ratio adjustment, alternative selection pressures, and the evolution of temperature-dependent sex determination.     

Maternal condition and offspring sex ratio in polygynous ungulates: a case study of bighorn sheep

The Trivers and Willard model (TWM) predicts that for sexuallydimorphic polygynous mammals, mothers able to provide a highlevel of care should bias offspring sex ratio in favor of sons.Contradictory results of empirical studies, however, suggestthat selective pressures for adaptive offspring sex ratio varywith species and environmental conditions. We report the resultsof a 29-year study of marked bighorn sheep (Ovis canadensis)in a population that underwent wide changes in density and wheremost females were weighed each year. Lamb sex ratio was independentof absolute ewe mass and yearly deviations from individual orpopulation average mass, but there was a nonsignificant trendtowards fewer males being born at high population density. Bighornsheep satisfy all the assumptions of the TWM but not its prediction:lamb sex ratio is independent of maternal ability to providecare. Recent hypotheses to explain the lack of relationshipbetween maternal condition and offspring sex in ungulates areunlikely to apply to bighorn sheep. We suggest that the TWMmay only apply when social rank strongly affects the abilityto provide maternal care. Those circumstances are likely tooccur for only a few species and within a narrow range of environmentalconditions.     

Newborns of Preeclamptic Women Show Evidence of Sex-Specific Disparity in Fetal Growth

BackgroundEvidence suggests that in response to in utero insults, male versus female infants have greater disadvantages in pregnancy outcome. In addition, there is a growing body of evidence suggesting that there is a sex-specific fetal response to maternal disease during pregnancy. We considered that a sex-specific relationship may exist between preeclampsia and reduced fetal growth.ObjectiveWe investigated if the relationship between preeclampsia and fetal growth was modified by fetal sex.MethodsWe limited the study population to singleton pregnancies of black and white normotensive and preeeclamptic women enrolled in the Collaborative Perinatal Project (1959–1965). The patients were offspring of 516 preeclamptic and 8801 normotensive women. After adjustment for confounders, interaction terms between preeclamptic status and fetal sex were evaluated to determine if the influence of preeclampsia on fetal growth varied with fetal sex. Separate linear and logistic regression models were then fitted for males and females to report the estimate of the relationship between preeclampsia and fetal growth by fetal sex. The results were stratified by preterm status (<37 vs ≥37 completed weeks of gestation). The mean head and chest circumferences, birthweight, ponderal index, and frequency of small for gestational age were examined. A 2-sided P value of <0.05 was considered statistically significant.ResultsThe results were stratified by preterm status. Male preterm offspring of preeclamptic mothers had greater reductions in chest circumference, head circumference, and birthweight than preterm female offspring of preeclamptic women (P = 0.01, P = 0.02, and P = 0.05, respectively, for interaction). Female versus male preterm offspring exposed to preeclampsia were less susceptible to being small for gestational age (synergy index 0.38; 95% CI, 0.00–0.84). The influence of preeclampsia on the growth of term offspring was more modest, and the influence of sex was opposite that in preterm infants. Compared with term offspring of normotensive women, the reduction in mean ponderal index was greater for female versus term male offspring of preeclamptic women (P = 0.02, interaction).ConclusionFetal growth was more impaired among male versus female preterm infants born to preeclamptic women. Our study underlined the importance of incorporating sex differences in the study of biological mechanisms for immediate- and long-term consequences of suboptimal fetal growth.     

Offspring are lighter at birth and smaller in adulthood when born after a brother versus a sister in humans

In mammals, including humans, it is more costly to produce sons than it is to produce daughters, with maternal survival and subsequent reproductive success diminished more by producing male over female offspring. It is therefore predicted that offspring who are produced by mothers who have previously produced sons versus daughters will be compromised by the relatively high cost their mother incurred in the previous reproductive episode. Such effects are potentially important because characters that determine offspring survival and fecundity ultimately contribute to maternal fitness. Using questionnaire-based data from a contemporary human population, I show that birthweight (irrespective of their sex) is lower in individuals born after an elder brother than in those born after an elder sister. In addition, I show that both men and women who were born after a male versus a female sibling have reduced adulthood height, a known correlate of reproductive success in both sexes. The results suggest that producing sons may have a negative effect on the fitness of subsequent offspring, which has implications for calculations of maternal fitness and for optimal sex allocation.